The lateral border recycling compartment (LBRC) is a recently-discovered membrane compartment located along the borders of vascular endothelial cells. Membrane from the LBRC recycles constitutively and rapidly between the compartment and the plasma membrane at borders between endothelial cells. The physiologic function of this constitutive recycling is not known. However, during leukocyte transmigration, recycling membrane from the LBRC is redirected-targeted to the site at which the leukocyte engages the endothelial cell junctions. It surrounds the leukocyte during the transmigration process providing increased membrane surface area in the junction and several key adhesion molecules that regulate transmigration. Blocking targeted recycling blocks leukocyte transmigration. Hence, the LBRC and its targeted recycling are critical factors regulating leukocyte transmigration in inflammation. Understanding how targeted recycling is regulated would provide insights into the control of the inflammatory response and potentially identify new therapeutic targets. Knowing the composition of the LBRC would allow us to formulate testable hypotheses about the function(s) of constitutive recycling and the regulation of targeted recycling. However, only three components of the LBRC are known: Platelet/endothelial cell adhesion molecule-1 (PECAM, CD31), CD99, and Junctional Adhesion Molecule A (JAM-A). In order to obtain an unbiased insight into the composition of the LBRC, we will isolate LBRC membrane from endothelial cell homogenates by density gradient centrifugation. We will analyze the protein composition of this membrane by two dimensional gel electrophoresis and identify membrane proteins unique to or enriched in the LBRC by comparison to the protein profiles of plasma membrane fractionated in parallel. Spots on the gels representing proteins that appear to be unique or markedly enriched in LBRC membrane will be excised, protease digested, and identified by mass spectrometry. As a complementary approach, a total proteomic comparison of LBRC and plasma membrane fractions will be performed. Candidate LBRC proteins will be validated in several ways. In the first step, we will localize these proteins in intact endothelial cells to be certain they have the correct distribution. They should be concentrated at the endothelial cell borders. Immunolocalization by confocal fluorescence microscopy will be performed. We expect most of the proteins will be well known and previously described. We will use existing antibodies against the candidate proteins where they are available and generate FLAG-tagged constructs of the candidate proteins, based on known sequences, where they are not. Those proteins that co-localize with authentic LBRC in intact cells will be further tested to determine whether they are truly components of the LBRC in functional assays. We will determine, using well-established assays in our lab, whether they recycle constitutively in the same manner as LBRC and participate in targeted recycling during leukocyte transmigration. Public Health Relevance: Most diseases (including atherosclerosis, asthma, and autoimmune diseases) involve an inflammatory response that is uncontrolled or misdirected. We have discovered a novel membrane compartment in endothelial cells (the cells that line blood vessels) that is critical for the inflammatory response. We will isolate this membrane compartment and identify its component proteins in order to understand how it functions and design better anti-inflammatory therapies.